3-Functionalised benzenesulphonamide based 1,3,4-oxadiazoles as selective carbonic anhydrase XIII inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2021.127856

A new series of benzenesulphonamide linked-1,3,4-oxadiazole hybrids (6a–s) has been synthesized and tested for their carbonic anhydrase inhibition against human (h) carbonic anhydrase (CA) isoforms hCA I, II, IX, and XIII. Fluorescence properties of some of the synthesized molecules were studied. Most of the molecules exhibited significant inhibitory power, comparable or better than the standard drug acetazolamide (AAZ) on hCA XIII. Out of 19 tested molecules, compound 6e (75.8 nM) was 3 times more potent than AAZ (250.0 nM) against hCA I, whereas compound 6e (15.4 nM), 6g (16.2 nM), 6h (16.4 nM) and 6i (17.0 nM) were found to be more potent than AAZ (17.0 nM) against isoform hCA XIII. It is anticipated that these compounds could be taken as the potential leads for the development of selective hCA XIII isoform inhibitors with improved potency.

Full text of DOI:10.1016/j.bmcl.2021.127856

Bioorg. Med. Chem. Lett. 37 (2021) 127856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Functionalised benzenesulphonamide based 1,3,4-oxadiazoles as
selective carbonic anhydrase XIII inhibitors: Design, synthesis and
biological evaluation
Baijayantimala Swaina, Abhaya, Priti Singha, Andrea Angelib, Kamtam Aashrithaa,
,
,c,*
Narayana Nageshd, Claudiu T. Supuranb *, Mohammed Arifuddina
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India
b
`
Universita degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
c Department of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally, Hyderabad 500001, T.S., India
d Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of benzenesulphonamide linked-1,3,4-oxadiazole hybrids (6a–s) has been synthesized and tested for
their carbonic anhydrase inhibition against human (h) carbonic anhydrase (CA) isoforms hCA I, II, IX, and XIII.
Fluorescence properties of some of the synthesized molecules were studied. Most of the molecules exhibited
significant inhibitory power, comparable or better than the standard drug acetazolamide (AAZ) on hCA XIII. Out
of 19 tested molecules, compound 6e (75.8 nM) was 3 times more potent than AAZ (250.0 nM) against hCA I,
whereas compound 6e (15.4 nM), 6g (16.2 nM), 6h (16.4 nM) and 6i (17.0 nM) were found to be more potent
than AAZ (17.0 nM) against isoform hCA XIII. It is anticipated that these compounds could be taken as the
potential leads for the development of selective hCA XIII isoform inhibitors with improved potency.
1,3,4-Oxadiazole
Carbonic anhydrase
hCA XIII isoform
Isoform selective inhibitors
Fluorescence
Carbonic anhydrases (CAs, EC 4.2.1.1) are the superfamily of met-
hydroxide derivative (L3-M2+-OHꢀ ) of the enzyme, that act as a strong
nucleophile (at neutral pH) on the CO2 molecule bound in a hydro-
phobic pocket. The water coordinated to the metal ion, which is found at
the bottom of the active site cavity generated this metal hydroxide
species.7 In addition, CAs also catalyze several other reactions, such as
the aldehyde hydration to gem-diols, the hydration of cyanate to car-
bamic acid, or cyanamide to urea and the hydrolysis of carboxylic,
sulphonic or phosphoric acids esters.8 Basically there are two main
classes of compounds that inhibit CAs: firstly, the metal complexing
inorganic anions (e.g., cyanide, cyanate, thiocyanate, azide, hydro-
gensulphide etc.), which were important for understanding the catalytic
and inhibitory mechanisms and secondly, the unsubstituted sulphona-
mides with the general formula RSO2NH2 (R = aryl; hetaryl; perha-
loalkyl) that led to the development of several classes of
alloenzymes with eight distinct families α, β, γ, δ, ζ, η, θ and ι, which are
found in all life kingdoms from eukaryotes to prokaryotes.1,2 The
α-CAs
are expressed in humans and are subdivided into 16 different isoforms
that vary by localization and catalytic activity: CA I, CA II, CA III, CA VII,
CA XIII are cytosolic; CA IV, CA IX, CA XII, CA XIV, CA XV membrane-
bound; CA VA and CA VB mitochondrial; and CA VI secreted in saliva
and colostrum. On the other hand, CA VIII, CA X, and CA XI are three
catalytically inactive forms referred to as CA-related proteins
(CARPs).3,4 These enzymes catalyze the reversible inter-conversion of
CO2 and H2O to bicarbonate and proton, for which, comparatively high
amounts of CAs are present in different tissues of most studied organ-
isms. They are associated with vital processes like respiration and
transport of CO2/bicarbonate between metabolizing tissues and the
lungs, pH and CO2 homeostasis, electrolyte secretion in a variety of
tissues/organs, various biosynthetic reactions such as the gluconeo-
genesis, lipogenesis, and ureagenesis, bone resorption, calcification,
tumorigenicity and many other physiologic or pathologic processes.5,6
The catalytically active species in all classes of CA, is a metal
pharmacological agents.9 Out of the 16 isoforms of
α CA, the cytosolic
CA XIII was reported in 2000s and shows similar catalytic activity to that
of mitochondrial isoenzyme CA V and cytosolic isoenzyme CA I, II, III.
This enzyme is expressed both in mouse (mCA XIII) and several human
(hCA XIII) tissues including salivary glands, kidney, small intestine,
* Corresponding authors at: Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad
500037, India (M. Arifuddin).
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), arifabib@gmail.com (M. Arifuddin).
https://doi.org/10.1016/j.bmcl.2021.127856
Received 2 December 2020; Received in revised form 27 January 2021; Accepted 5 February 2021
Available online 18 February 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

B. Swain et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127856
Fig. 1. Representative examples of benzenesulphonamide and 1,3,4-oxadiazole as carbonic anhydrase inhibitors and rationale for designed molecules (6a–s).
colon, uterus and testis. These findings suggest that the widespread
distribution of hCA XIII leads to the development of inhibitors for
treatment and prevention of various disorders.10–14
investigations in the design of novel CA inhibitors containing oxadiazole
moieties linked to 3-functionalised benzene sulphonamide. The syn-
thesis of the target compounds start with reaction of commercially
available benzoic acids (1a–c) and chlorosulphonic acid were reacted at
110 ◦C to afford the 3-(chlorosulphonyl)benzoic acids (2a–d) which
were in the second step treated with ammonium hydroxide solution at
However, substitution on the tail part of benzenesulphonamide with
various heterocyclic or aromatic rings enhance the inhibitory activity by
modifying absorption and gastro-intestinal tolerance. Dichlorphena-
mide, celecoxib, sulpiride, valdecoxib, indisulam (Fig. 1a–e) are some of
the most popular marketed drug which contains the benzenesulphona-
mide moiety.15 However, the heterocycles with 1,3,4-oxadiazole moi-
eties are also exhibiting wide range of biological activities that included
anticancer, antibacterial, antifungal, analgesic, anti-inflammatory,
anticonvulsant, antihypertensive, antiviral, anti HIV, and antidiabetic
properties.16 Sharma et al. have reported that 1,3,4-oxadiazole benze-
nesulphonamide hybrids showed potential inhibition against hCA I, II,
IX (Fig. 1f).17 Bianco et al. have reported selectivity of N-acylbenzene-
sulphonamide dihydro-1,3,4-oxadiazole hybrids against hCA IX and XII
(Fig. 1g).18 In view of the importance of these scaffold in the present
work, we designed and synthesized a series of new molecules with 3-
functionalised benzenesulphonamide incorporated 1,3,4-oxadiazoles
which could inhibit different CAs isoforms with improved selectivity.
Considering the inhibitory potency of the 3-functionalised benzene
sulphonamide from our previous paper, here we extend our
0
◦C to give the corresponding 3-sulphamoylbenzoic acids (3a–c).
Compounds 3a–c were subjected to esterification with SOCl2 and MeOH
forming 4a–c, followed by hydrazide formation 5a–c using
NH2NH2⋅H2O under reflux. Finally, the hydrazides under-
went transition-metal-free oxidative cyclization forming 1,3,4-oxadia-
zoles 6a–s in the presence of potassium carbonate, molecular I2 with
different aromatic aldehydes.
Reagent and reaction conditions: i) HSO3Cl (5 equiv.), 110 ◦C,
6–8 h, 70–80%; ii) NH4OH sol., 0 ◦C, 2 h; iii) MeOH, SOCl2, reflux,
overnight, 90–98%; iv) NH2NH2⋅H2O (3 equiv.), EtOH, reflux, 4 h,
75–80%; v) Substituted benzaldehyde (1 equiv.), I2 (1.2 equiv.), K2CO3
(3 equiv.), DMSO, 100 ◦C, N2, reflux, 74–80%.
The target benzenesulphonamide linked-1,3,4-oxadiazole hybrids
(6a–s) were screened for their potentials against four different hCA
isoforms, namely, hCA I, II, XIII (the cytosolic) and hCA IX, (the tumor-
associated) by a stopped-flow CO2 hydrase assay, taking acetazolamide
2

B. Swain et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127856
Table 1
Inhibition of hCA isoforms I, II, IX and XIII with target compounds (6a–s) and acetazolamide (AAZ) as a standard drug (Ki = nM).*
Compound
Structure
hCA I
hCAII
hCA IX
hCA XIII
6a
629.3 ± 51.1
288.6 ± 16.8
108.2 ± 4.5
30.9 ± 1.7
6b
6c
754.7 ± 46.5
2568 ± 138
1550 ± 153
82.2 ± 5.9
76.8 ± 7.2
36.7 ± 2.4
31.4 ± 1.5
833.5 ± 61.7
6d
6e
1392 ± 131
75.8 ± 5.6
904.9 ± 72.8
89.8 ± 6.1
68.3 ± 5.6
76.3 ± 4.2
38.5 ± 3.2
15.4 ± 0.7
6f
861.6 ± 64.4
5023 ± 482
52.4 ± 2.2
44.0 ± 2.9
57.1 ± 3.3
19.1 ± 1.6
16.2 ± 1.1
6g
285.9 ± 26.6
6h
6i
915.9 ± 78.9
887.9 ± 77.2
608.0 ± 36.2
407.5 ± 32.1
54.5 ± 4.5
76.3 ± 4.8
16.4 ± 0.9
17.0 ± 1.1
6j
268.3 ± 18.5
217.3 ± 13.7
85.3 ± 4.0
18.4 ± 0.9
6k
6l
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
6m
6n
6o
6p
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
(continued on next page)
3

B. Swain et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127856
Table 1 (continued)
Compound
Structure
hCA I
hCAII
hCA IX
hCA XIII
6q
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
6r
6s
>10,000
>10,000
>10,000
>10,000
AAZ
250.0
12.1
25.8
17.0
*
Mean from 3 different assays, by a stopped flow technique (from 3 different determinations).
(AAZ), as a standard drug. The following Structure-Activity Relationship
(SAR) can be inferred from the inhibition data of benzenesulphonamide
linked ꢀ 1,3,4-oxadiazole hybrids (6a–s) as shown in Table 1:
were moderate to weak inhibitors of hCA II with Ki ranging 52.4 nM-
1.5 µM and hCA IX with Ki ranging 44.0 nM- 0.10 µM while com-
pounds with substitution (R1) on benzenesulphonamide ring (6k–s)
were ineffective and exhibited inhibitory potencies with Ki > 10 µM
as compared to standard AAZ for both hCA II (Ki = 12.1 nM) and IX
(Ki = 25.8 nM).
• The target compounds 6a–s had shown weak inhibition towards the
cytosolic isoform hCA I except one molecule having an excellent
inhibition potency that was 6e with Ki = 75.8 nM as compared to
AAZ (Ki = 250 nM). However, the compounds without substitution
on the benzenesulphonamide ring (6a–j) have shown moderate in-
hibition with Ki ranging 0.268 µM–5.0 µM, whereas the compounds
with substitution on the benzenesulphonamide ring (6k–s) were
ineffective towards the isoform (Ki > 10000 nM).
• The molecules investigated for the inhibition of the new isoenzyme
CA XIII were quite excellent. Out of total nineteen molecules ten
(6a–j) were showing very good inhibitory potencies with Ki ranging
15.4–38.5 nM as compared to AAZ (Ki = 17.0 nM). Compounds 6e–j
where R2 was substituted with electron withdrawing group (–NO2,
–Cl, –CN) and heteroaromatic ring (–thiophene) have shown better
activity than that of the molecules (6a–d) with R2 containing elec-
tron donating group (–H, –OMe) and in all these R1 was without any
substitution. Similarly, the molecules (6k–s) have not shown any
inhibition because of the substitution on benzenesulphonamide ring.
• All the synthesized compounds (6a–s) were less potent than the
standard drug AAZ for both cytosolic isoform hCA II and the trans-
membrane tumor-associated isoform hCA IX. Here also the com-
pounds without substitution on benzenesulphonamide ring (6a–j)
Fig. 2. SAR for the target CAIs toward hCA I and XIII isoforms.
4

B. Swain et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127856
Fig. 3. Fluorescence emission spectra of compounds 6b, 6c, and 6d in DMSO.
• The inhibition order of the four most potent benzenesulphonamide
linked-1,3,4-oxadiazole hybrids are as follows, hCA XIII inhibition:
(6e) > (6g) > (6h) > (6i). Most active molecule 6e has shown very
good inhibition against both isoforms hCA I (75.8 nM) and hCA XIII
(15.4 nM).
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
• Furthermore, it is interesting to note here that when the molecules
contain any substitution (R1) on the benzenesulphonamide ring
those molecules (6k–s) were devoid of any inhibitory activity against
all the four isoforms with Ki > 10000 nM. Again the potent molecules
were more selective towards hCA XIII over hCA I. The obtained SAR
toward hCA I and XIII isoforms was illustrated in Fig. 2.
The authors BS, A, PS and KA are thankful to Department of Phar-
maceuticals, Ministry of Chemicals and Fertilizers, Govt. of India, New
Delhi for the award of NIPER fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127856.
Fluorescence studies were performed for the synthesized molecules
(6a–s) as 1,3,4-oxadiazoles are well known for exhibiting fluorescence
phenopmena.19 The molecules were diluted in DMSO and the excitation
and emission spectra were investigated. Out of 19 molecules the exci-
tation and emission spectra of three compounds 6b, 6c and 6d showed
prominent shifts to longer wavelengths, in contrast to the other cases in
which the shifts were typically only marginal. Fig. 3. shows the fluo-
rescence emission spectra of compounds 6b, 6c and 6d. Fluorescence
properties of these compounds suggest that they may hold a potential for
applications as chemical probes.
References
1 Swain B, Angeli A, Angapelly S, et al. J Enzyme Inhib Med Chem. 2019;34:1199–1209.
2 (a) Supuran CT. J Enzyme Inhib Med Chem. 2016;31:345–360.(b) Nocentini A,
Supuran CT. Expert Opin Drug Discov. 2019;14:1175–1197.
3 Lomelino CL, Supuran CT, Kenna RM. Int J Mol Sci. 2016;17:1150–1162.
4 Swain B, Digwal CS, Angeli A, et al. Bioorg Med Chem. 2019;27, 115090.
5 Tuccinardi T, Bertini S, Granchi C, et al. Bioorg Med Chem. 2013;21:1511–1515.
6 Mishra CB, Kumari S, Angeli A, et al. J Enzyme Inhib Med Chem. 2016;31:174–179.
7 Supuran CT. J Enzyme Inhib Med Chem. 2012;27:759–772.
8 Alterio V, Fiore AD, Ambrosio KD, Supuran CT, Simone GD. Chem Rev. 2012;112:
4421–4468.
In summary, the current research work explained the design and
synthesis of new benzenesulphonamide linked-1,3,4-oxadiazole hybrids
(6a–s) as significant human carbonic anhydrase inhibitors. The newly
synthesized molecules in variable degrees of inhibition affected all the
tested CA isoforms (hCA I, II, IX, XIII). Some of the synthesiz
9 Supuran CT, Scozzafava A. Exp Opin Ther Patents. 2000;10:575–600.
10 Lehtonen J, Shen B, Vihinen M, et al. J Biol Chem. 2004;279:2719–2727.
11 Innocenti A, Lehtonen JM, Parkkila S, Scozzafavaa A, Supuran CT. Bioorg Med Chem
Lett. 2004;14:5435–5439.
12 Lehtonen JM, Parkkila S, Vullo D, Casini A, Scozzafava A, Supuran CT. Bioorg Med
Chem Lett. 2004;14:3757–3762.
ed molecules exhibited fluorescence properties, which specify their
potential usefulness in the field of material chemistry. The present target
sulfonamide molecules are selectivity as most of them showed good
selectivity toward hCA XIII (Ki ranges 15.4–38.5 nM) over CA I (Ki
ranges 75.8–5023 nM). SAR results revealed that incorporation of sub-
stitution (R1) on the benzenesulphonamide ring (6k–s) led to a lack of
effectiveness against all the tested isoforms (Ki > 10,000 nM); whereas,
molecules with substitution R2 on the tail part containing 1,3,4-oxadia-
zole and no substitution on head part (R1 ¼ H) led to a better inhibition
with Ki ranging: 268.3 nM–0.5 µM for hCA I, 52.4 nM–1.5 µM for hCA II,
44.0 nM–0.10 µM for hCA IX and 15.4 nM–38.5 nM hCA XIII. Further-
more, molecules with R2 containing electron withdrawing group (–NO2,
–Cl, –CN) and heteroaromatic ring (–furan) (6e–j) were more potent
inhibitory action than those containing electron donating group (6a–d)
against hCA XIII. In addition, molecule 6e is the most potent one
compared to the standard AAZ both in case of hCA I (Ki = 75.8 nM) and
hCA XIII (Ki = 15.4 nM). Hence, it is anticipated that the molecules
which are more potent could be taken as the lead for the design and
development of selective hCA XIII inhibitor with better inhibition
potency.
13 Hilvo M, Supuran CT, Parkkila S. Curr Top Med Chem. 2007;7:893–899.
¨
¨ ¨
¨
14 Kummola L, Hamalainen JM, Kivela J, et al. BMC Cancer. 2005;5:41.
15 Supuran CT. Nat Rev Drug Discov. 2008;7:168–181.
16 Bala S, Kamboj S, Kumar A. J Pharm Res. 2010;3:2993–2997.
17 Sharma V, Kumar R, Angeli A, Supuran CT, Sharma PK. Eur J Med Chem. 2020;193,
112219.
18 Bianco G, Meleddu R, Distinto S, et al. ACS Med Chem Lett. 2017;8:792–796.
19 Yan YN, Pan W, Song HC. Dyes Pigm. 2010;86:249–258.
20 Khalifah RG. J Biol Chem 1971, 246, 2561–2573. An SX.18MV-R applied
photophysics (Oxford, UK) stopped-flow instrument has been used to assay the
catalytic/inhibition of various CA isozymes.20 Phenol Red (at a concentration of 0.2
mM) has been used as indicator, working at the absorbance maximum of 557 nm,
with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining
constant the ionic strength; these anions are not inhibitory in the used
concentration), following the CA-catalyzed CO2 hydration reaction for a period of
5–10 s. Saturated CO2 solutions in water at 25 ◦C were used as substrate. Stock
solutions of inhibitors were prepared at a concentration of 10 μM (in DMSO-water 1:
1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At
least 7 different inhibitor concentrations have been used for measuring the inhibition
constant. Inhibitor and enzyme solutions were preincubated together for 10 min at
room temperature prior to assay, in order to allow for the formation of the E-I
complex. Triplicate experiments were done for each inhibitor concentration, and the
values reported throughout the paper are the mean of such results. The inhibition
constants were obtained by non-linear least-squares methods using the Cheng-
Prusoff equation, as reported earlier,21 and represent the mean from at least three
different determinations. All CA isozymes used here were recombinant proteins
obtained as reported earlier by our group.22,23 Their concentrations in the assay
system were in the range of 4.5–12.3 nM
Declaration of Competing Interest
The authors declare that they have no known competing financial
5

B. Swain et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127856
21 Draghici B, Vullo D, Akocak S, Walker EA, Supuran CT, Ilies MA. Chem Commun.
2014;50:5980–5983.
23 Akocak S, Lolak N, Vullo D, Durgun M, Supuran CT. J Enzyme Inhib Med Chem. 2017;
32:1305–1312.
22 Ilies MA, Vullo D, Pastorek J, et al. J Med Chem. 2003;46:2187–2196.
6